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Identification of HIF-2?-regulated genes that play a role in human microvascular endothelial sprouting during prolonged hypoxia in vitro.

ABSTRACT: During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1? and HIF-2?. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2? partially restores sprout formation. The present study identifies novel HIF-2?-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile of primary human microvascular endothelial cells (hMVECs) that were cultured at 20 % oxygen was compared to hMVECs that were cultured at 1 % oxygen for 14 days by using genome-wide RNA-sequencing. The differentially regulated genes in hypoxia were compared to the genes that were differentially regulated upon silencing of HIF-2? in hypoxia. Surprisingly, KEGG pathway analysis showed that metabolic pathways were enriched within genes upregulated in response to hypoxia and enriched within genes downregulated upon HIF-2? silencing. Moreover, 51 HIF-2?-regulated genes were screened for their role in endothelial sprouting in hypoxia, of which four genes ARRDC3, MME, PPARG and RALGPS2 directly influenced endothelial sprouting during prolonged hypoxic culturing. The manipulation of specific downstream targets of HIF-2? provides a new, but to be further evaluated, perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization of implanted tissue-engineered scaffolds.

SUBMITTER: Nauta TD

PROVIDER: S-EPMC5306362 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Identification of HIF-2α-regulated genes that play a role in human microvascular endothelial sprouting during prolonged hypoxia in vitro.

Nauta Tessa D TD van den Broek Marloes M Gibbs Sue S van der Pouw-Kraan Tineke C T M TC Oudejans Cees B CB van Hinsbergh Victor W M VW Koolwijk Pieter P

Angiogenesis 20161003 1

During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2α partially restores sprout formation. The present study identifies novel HIF-2α-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile ...[more]

PMID: 27699500

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Project description:BackgroundAstrocytes (AC) are essential for brain homeostasis. Much data suggests that AC support and protect the vascular endothelium, but increasing evidence indicates that during injury conditions they may lose their supportive role resulting in endothelial cell activation and BBB disturbance. Understanding the triggers that flip this switch would provide invaluable information for designing new targets to modulate the brain vascular compartment. Hypoxia-inducible factor-1 (HIF-1) has long been assumed to be a culprit for barrier dysfunction as a number of its target genes are potent angiogenic factors. Indeed AC themselves, reservoirs of an array of different growth factors and molecules, are frequently assumed to be the source of such molecules although direct supporting evidence is yet to be published. Being well known reservoirs of HIF-1 dependent angiogenic molecules, we asked if AC HIF-1 dependent paracrine signaling drives brain EC disturbance during hypoxia.MethodsFirst we collected conditioned media from control and siRNA-mediated HIF-1 knockdown primary rat AC that had been exposed to normoxic or hypoxic conditions. The conditioned media was then used to culture normoxic and hypoxic (1% O2) rat brain microvascular EC (RBE4) for 6 and 24 h. Various activation parameters including migration, proliferation and cell cycling were assessed and compared to untreated controls. In addition, tight junction localization and barrier stability per se (via permeability assay) was evaluated.ResultsAC conditioned media maintained both normoxic and hypoxic EC in a quiescent state by suppressing EC metabolic activity and proliferation. By FACs we observed reduced cell cycling with an increased number of cells in G0 phase and reduced cell numbers in M phase compared to controls. EC migration was also blocked by AC conditioned media and in correlation hypoxic tight junction organization and barrier functionality was improved. Surprisingly however, AC HIF-1 deletion did not impact EC responses or barrier stability during hypoxia.ConclusionsThis study demonstrates that AC HIF-1 dependent paracrine signaling does not contribute to AC modulation of EC barrier function under normoxic or hypoxic conditions. Thus other cell types likely mediate EC permeability in stress scenarios. Our data does however highlight the continuous protective effect of AC on the barrier endothelium. Exploring these protective mechanisms in more detail will provide essential insight into ways to prevent barrier disturbance during injury and disease.

Dataset Information

Identification of HIF-2?-regulated genes that play a role in human microvascular endothelial sprouting during prolonged hypoxia in vitro.

Publications

Identification of HIF-2α-regulated genes that play a role in human microvascular endothelial sprouting during prolonged hypoxia in vitro.

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